JP5697123B2 - Method for introducing nucleic acid into cells using acidified polyethylenimine - Google Patents

Description

  The present invention relates to a method for introducing a nucleic acid into a cell using acidified polyethyleneimine. More specifically, the present invention relates to a method for introducing a nucleic acid into a cell, comprising mixing acidified polyethyleneimine and a nucleic acid under acidic conditions, and treating the cell with the resulting mixture. The present invention also relates to a reagent for introducing a nucleic acid into cells comprising an acidified polyethyleneimine solution and a reagent kit containing the solution.

  Introduction of nucleic acids into cells is essential for basic research in molecular cell biology as well as medical applications such as gene therapy. For example, transfection methods using viral vectors and non-viral transfection methods are known as methods for introducing genes into cells.

  Transfection methods utilizing viral vectors result in high levels of gene transfer. Viral vectors are efficient gene carriers, but their use is limited due to the induction of immune responses and virus-related pathogenicity.

  Non-viral transfection methods do not have the problems of transfection methods using viral vectors and contribute to the creation of pharmaceuticals from nucleic acids (Non-patent Document 1). According to this method, the product can be produced in large quantities at an acceptable price with high reproducibility, and can be stored stably for a long period of time.

  Many non-viral transfection methods are known, and the main method is exemplified by a method using a cationic lipid and / or a cationic polymer. Currently, a series of non-viral transfection reagents based on cationic lipids and cationic polymers are commercially available and used. However, in the transfection method using such a reagent, when the nucleic acid is taken into the cell together with the reagent by phagocytosis, most of the nucleic acid is decomposed together with the reagent by lysosome, resulting in low transfection efficiency. There is. Also, since many of the commercially available transfection reagents are currently quite expensive, their high cost is problematic for experiments involving many and / or large scale transfections.

  Polyethyleneimine (hereinafter abbreviated as PEI), a cationic polymer, is a low-cost, non-viral, non-liposomal transfection reagent and is known as one of the most cost-effective media. PEI has a high cationic charge density potential and efficiently compacts DNA to form a stable complex called a polyplex. Since polyplex is taken up into cells by endocytosis, transfection is efficiently performed by using PEI (Non-patent Document 2). In addition, PEI has a high pH buffering capacity, protects incorporated DNA from degradation by lysosomes, and efficiently releases lysosomes to the cytoplasm. Thus, PEI is useful as a transfection reagent and is used in vitro and in vivo for transfection of mammalian cells with DNA (Non-patent Documents 2-4).

  However, transfection methods using PEI have problems such as considerable toxicity of PEI and a significant decrease in transfection efficiency due to the low stability of PEI.

  Extensive research has been conducted on PEI size, structure, and chemical modifications to improve PEI transfection efficiency (4-11) and PEI / DNA polyplexes or condensed DNA It has also been studied to add an adjuvant to the non-patent document 12-13.

Special table 2008-509076. Special table 2006-516259.

Davis, M.E. (2002) Non-viral gene delivery systems. Curr. Opin. Biotechnol. 13, 128-131. Boussif, O. et al., (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine.Proc. Natl. Acad. Sci. USA 92, 7297-7301. Abdallah, B. et al., (1996) A powerful nonviral vector for in vivo gene transfer into the adult mammalian brain: Polyethylenimine.Hum. Gene Ther. 7, 1947-1954. Goula, D. et al., (1998) Size, diffusibility and transfection performance of linear PEI / DNA complexes in the mouse central nervous system.Gene Ther. 5, 712-717. Zanta, M.A. et al., (1997) In vitro gene delivery to hepatocytes with galactosylated polyethylenimine. Bioconjug. Chem. 8, 839-844. Fischer, D. et al., (1999) A novel non-viral vector for DNA delivery based on low molecular weight, branched polyethylenimine: Effect of molecular weight on transfection efficiency and cytotoxicity. Pharm. Res. 16, 1273-1279. Ogris, M. et al., (1999) PEGylated DNA / transferrin-PEI complexes: Reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery.Gene Ther. 6, 595-605. Wightman, L. et al., (2001) Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo.J. Gene Med. 3, 362-372. Durocher, Y. et al., (2002) High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res. 30, e9. Brissault, B. et al., (2003) Synthesis of linear polyethylenimine derivatives for DNA transfection. Bioconjug. Chem. 14, 581-587. Thomas, M. et al., (2005) Full deacylation of polyethylenimine incremental boosts its gene delivery efficiency and specificity to mouse lung.Proc. Natl. Acad. Sci. USA 102, 5679-5684. Ogris, M. et al., (1998) The size of DNA / transferring-PEI complexes is an important factor for gene expression in cultured cells.Gene Ther. 5, 1425-1433. Tseng, W.C. et al., (2007) Trehalose enhances transgene expression mediated by DNA-PEI complexes. Biotechnol. Prog. 23, 1297-1304.

  Studies aimed at improving the transfection efficiency of PEI have focused on PEI size, structure, and chemical modification. However, increasing the transfection efficiency of PEI and reducing its toxicity has not yet been fully achieved.

  The object of the present invention is to find a method for achieving an increase in the transfection efficiency of PEI and a reduction in its toxicity, to provide a PEI with a high transfection efficiency, and further to an efficient cell using such a PEI. It is to provide a method for introducing a nucleic acid.

  The present inventors have intensively studied to solve the above problems. In this study, in order to overcome the disadvantages of PEI, the conditions for PEI solution and polyplex formation, as well as the transfection period, were examined. As a result, it was found that high transfection efficiency can be secured by acidification of PEI and polyplex formation under acidic conditions. Furthermore, it was found that by removing PEI after transfection, sufficient transfection efficiency and toxicity reduction can be achieved. The present inventors have achieved the present invention from these findings.

That is, the present invention relates to:
1. A method for introducing nucleic acid into a cell, including the following steps:
(1) mixing polyethyleneimine (PEI) and nucleic acid under acidic conditions of pH 3.5 to pH 4.5, and (2) treating cells with the resulting mixture,
2. The method for introducing a nucleic acid into a cell according to the above 1. further comprising the following steps following the step (2):
(3) replacing the cell culture medium with fresh culture medium;
3. The method for introducing a nucleic acid into the cell according to 1 or 2 above, wherein PEI is linear PEI,
4. A method for introducing a nucleic acid into a cell according to 1 or 2 above, wherein the PEI is a linear PEI having a molecular weight of about 25,000.
5. The method for introducing a nucleic acid into a cell according to any one of 1 to 4 above, wherein the weight ratio of PEI to the nucleic acid is 4: 1 to 8: 1.
6. The method for introducing a nucleic acid into the cell according to any one of 1 to 5 above, wherein the PEI according to any one of 1 to 5 is a solution having a pH of 0.5 to pH 2.0 (PEI solution),
7. The method for introducing a nucleic acid into the cell according to any one of 1 to 5 above, wherein the PEI according to any one of 1 to 5 is a hydrochloric acid solution of PEI.
8. The method for introducing a nucleic acid into a cell according to any one of 1. to 5. above, wherein the PEI according to any one of 1. to 5. is a solution prepared by dissolving PEI in a 0.2N hydrochloric acid solution. ,
9. A method for introducing nucleic acid into a cell comprising the following steps:
(1) A step of mixing polyethyleneimine (PEI) and nucleic acid under acidic conditions of pH 3.5 to pH 4.5,
(2) treating the cells with the resulting mixture, and (3) replacing the cell culture medium with fresh culture medium,
Here, PEI is a solution prepared by dissolving linear PEI having a molecular weight of about 25,000 in 0.2N hydrochloric acid solution, and the weight ratio of PEI to nucleic acid is 4: 1 to 8: 1.
10. A reagent kit for cells containing a polyethyleneimine (PEI) solution, wherein the pH of the PEI solution is from 0.5 to 2.0,
11. The reagent kit for introducing a nucleic acid into a cell according to 10., wherein the PEI solution is a hydrochloric acid solution of PEI,
12. The reagent kit for introducing a nucleic acid into a cell according to 10., wherein the PEI solution is a solution prepared by dissolving PEI in a 0.2N hydrochloric acid solution,
13. The reagent kit for introducing a nucleic acid into a cell according to any one of 10. to 12. above, wherein the PEI contained in the PEI solution is linear PEI,
14. The reagent kit for introducing a nucleic acid into a cell according to any one of 10. to 12. above, wherein the PEI contained in the PEI solution is a linear PEI having a molecular weight of about 25,000,
15. A reagent kit containing a polyethyleneimine (PEI) solution, wherein the PEI solution is a solution prepared by dissolving linear PEI having a molecular weight of about 25,000 in 0.2N hydrochloric acid solution. Nucleic acid introduction reagent kit,
16. A reagent for introducing a nucleic acid into a cell comprising polyethyleneimine (PEI), wherein the pH of the PEI solution is from 0.5 to 2.0, and the introduction efficiency of the nucleic acid to a cell retaining the introduction efficiency is maintained.

  ADVANTAGE OF THE INVENTION According to this invention, the nucleic acid introduction | transduction method to a cell characterized by mixing PEI and a nucleic acid on acidic conditions, and processing a cell with the obtained mixture can be provided.

  Moreover, according to the present invention, a reagent for introducing a nucleic acid into a cell comprising an acidified PEI solution and a reagent kit for introducing a nucleic acid into a cell containing the solution can be provided.

  In the method for introducing nucleic acid into cells according to the present invention, high transfection efficiency and minimal toxicity can be achieved in introducing nucleic acid into various types of cells and cultured cells. In addition, the cost required for the present nucleic acid introduction method can be dramatically reduced to about 1: 10000 as compared with the nucleic acid introduction method using various commercially available reagents.

  In addition, the acidified PEI solution maintains the transfection activity of PEI even during long-term storage, as compared with the neutralized PEI solution, and thus can maintain the efficiency of introducing nucleic acid into cells.

  Thus, according to the present invention, nucleic acid introduction into cells can be achieved simply, cost-effectively and with high efficiency.

It is a figure which shows that the transfection efficiency by PEI / DNA polyplex increased by performing DNA condensation by PEI under acidic conditions. EGFP expression vector and PEI were mixed in HBS (pH 7.4) or LBS (pH 3.5) to cause DNA condensation (indicated as “Compact in HBS” or “Compact in LBS”). Transient transfection of HeLa cells was performed with the obtained PEI / DNA polyplex, and EGFP expression in the cells was analyzed by flow cytometry. Untreated cells were similarly analyzed (indicated as No treatment in the figure). In the figure, the vertical axis represents the cell number, and the horizontal axis represents the fluorescence intensity. In the figure, the shaded area shows a histogram of untreated cells, and the solid line area shows a histogram of transfected cells. Transient transfection efficiency was assessed by counting the number of cells expressing EGFP at high levels (thick arrows) and low to high levels (thin arrows). (Example 1) It is a figure which shows that the toxicity to the cell in the transfection by PEI / DNA polyplex fell by performing DNA condensation by PEI under acidic conditions. Transfections were performed as described in the description of Figure 1-A, then transfected HeLa cells were stained with PI and dead cells stained with PI were analyzed by flow cytometry. Data are expressed as mean ± standard deviation (n = 3). The vertical axis of the figure shows the ratio of dead cells (Dead cells (%)). (Example 1) It is a figure which shows the result of having examined the effect which PEI / DNA ratio and DNA condensation pH have on transfection efficiency. PEI and EGFP expression vectors are mixed in LBS (pH 3.5) at various PEI / DNA ratios, and the resulting PEI / DNA polyplex is transiently transfected with HeLa cells to flow EGFP expression and toxicity Analyzed by cytometry. Untreated cells were similarly analyzed (indicated as No treatment in the figure). The vertical axis in the figure indicates the percentage of cells (% Cells). The numbers in the figure indicate the PEI / DNA ratio at the time of preparation of the PEI / DNA polyplex used for transfection (Transfection [PEI / DNA ratio (μg / μg)]). In the figure, the black column indicates EGFP high expressing cells (Cells expressing high levels of EGFP), and the white column indicates dead cells. (Example 2) It is a figure which shows the result of having examined the effect which PEI / DNA ratio and DNA condensation pH have on transfection efficiency. PEI and EGFP expression vectors were mixed in LBS (pH 3.5) to give a PEI / DNA ratio of 6 (μg / μg) (indicated as PEI / EGFP ratio = 6 in the figure), and the resulting PEI / DNA HeLa cells were transiently transfected with polyplex and EGFP expression was analyzed by flow cytometry. Untreated cells were similarly analyzed (indicated as No treatment in the figure). In the figure, the vertical axis represents the cell number, and the horizontal axis represents the fluorescence intensity. In the figure, the shaded area shows a histogram of untreated cells, and the solid line area shows a histogram of transfected cells. Transfection efficiency was assessed by counting the number of cells expressing EGFP at high levels (thick arrows) and low to high levels (thin arrows). (Example 2) It is a figure which shows the result of having examined the effect which DNA condensation pH has on transfection efficiency. PEI and EGFP expression vectors were mixed in HBS (pH 7.4) or LBS (pH 3.5, 4.0, 4.5) to a ratio of 5 (μg / μg), and the resulting PEI / DNA polyplex was HeLa Transient transfection of cells was performed. The vertical axis in the figure indicates the FGFP increase in the number of cells expressing EGFP. The numerical value in the figure indicates the pH of the DNA condensation medium (pH of compaction medium). In the figure, the black column and the white column represent cells expressing EGFP at a high level (indicated as High level expression) and cells expressing low to high level (Low to high level expression), respectively. Data are expressed as mean ± standard deviation (n = 3). (Example 2) It is a figure which shows the result of having transfected COS-1 and HeLa S3 cell by the PEI / DNA polyplex obtained on acidic condition. EGFP expression was examined with a fluorescence microscope. Cells were transiently transfected with a polyplex formed by mixing 5 μg of EGFP expression vector and 25 μg of PEI in LBS (pH 4.0), and after 12 hours of culture, the medium was washed with fresh serum. The medium was exchanged. Cell morphology and EGFP expression were then observed with a Nomarski differential interference microscope and fluorescence microscope. Scale bars are 10μm. (Example 3) It is a figure which shows the result of having compared the transfection by the PEI / DNA polyplex obtained under acidic conditions, and the transfection using the optimal amount of TransIT-LT1 using COS-1 cells. Cells were transiently transfected with a polyplex formed by mixing 1 μg EGFP expression vector and 5 μg PEI in LBS (pH 4.0), or with an optimal amount of TransIT-LT1. Cell morphology and EGFP expression were then observed with a Nomarski differential interference microscope and fluorescence microscope. Scale bars are 10μm. (Example 3) It is a figure which shows the result of having performed the transfection of the Dami cell which carried out suspension culture using the PEI / DNA polyplex obtained on acidic conditions. Suspension cultured Dami cells were attached to culture dishes and transiently transfected with polyplexes formed by mixing 4 μg EGFP expression vector and 26 μg PEI in LBS (pH 4.0) . Cell morphology and EGFP expression were then observed with a Nomarski differential interference microscope and fluorescence microscope. Scale bars are 10μm. (Example 3) It is a figure which shows that it succeeded in the transfection of the HCT116 cell in which cell aggregation and cell death generate | occur | produce because it is highly sensitive to the contact with PEI using the PEI / DNA polyplex obtained under acidic conditions. Cells were transiently transfected once or twice with polyplexes formed by mixing 6 μg CD25 expression vector and 7.5 μg PEI in LBS (pH 4.0) (in the figure, After staining with CD25 transfection (once) or CD25 transfection (twice)) and anti-CD25 antibody, CD25 expression was analyzed with a cell sorter. Untreated cells were similarly analyzed (indicated as No treatment in the figure). In the figure, the vertical axis represents the number of HCT116 cells (Cell number (HCT116)), and the horizontal axis represents the fluorescence intensity. In the figure, the shaded area represents a histogram of untreated cells, and the solid line area represents cells transfected. Transfection efficiency was evaluated by counting the number of cells expressing CD25 at low to high levels (thin arrows). (Example 3) It is a figure which shows the result of having produced the stable transfection cell clone using the PEI / DNA polyplex obtained on acidic conditions. Stable transfection of HeLa S3 cells was performed using a polyplex formed by mixing an EGFP expression vector and PEI in LBS (shown as Stable transfection in the figure). Subsequently, zeocin resistant colonies were cloned in 2 weeks (indicated as HeLa S3 colonies in the figure), and then cell morphology and EGFP expression were observed with a Nomarski differential interference microscope and a fluorescence microscope. Scale bars are 10μm. It is a figure which shows the specific example of the nucleic acid introduction | transduction method to the cell which uses acidified PEI.

  The present invention relates to a method for introducing a nucleic acid into a cell using PEI. The method according to the present invention is characterized in that PEI and nucleic acid are mixed under acidic conditions, and cells are treated with the resulting mixture.

  The present invention also relates to a reagent for introducing a nucleic acid into a cell comprising an acidified PEI solution and a reagent kit for introducing a nucleic acid into a cell containing the solution. The PEI solution according to the present invention is a PEI solution dissolved in an acidic solvent.

  “Nucleic acid” is a nucleotide unit consisting of a purine or pyrimidine base, a sugar, and a phosphate, and this phosphate forms a diester bond between the 3 ′ and 5 ′ carbons of the sugar between each nucleotide. Refers to a chain-like polynucleotide polymerized by. Depending on the difference in sugar, deoxyribonucleic acid (DNA) whose sugar moiety is deoxyribose and ribonucleic acid (RNA) which is ribose are roughly classified.

  The term “nucleic acid” encompasses genes, DNA, RNA, oligonucleotides, and polynucleotides. The term “nucleic acid” is also used herein interchangeably with gene, DNA, RNA, oligonucleotide, and polynucleotide.

  The term “introducing nucleic acid into a cell” is used interchangeably herein with the term “transfection of a cell”.

  “Cells” include isolated cells, cells contained in isolated biological tissue, and cultured cells. When the cell is an isolated cell or a cultured cell, the cell may be an adherent cell or a suspension cell.

  PEI is a cationic polymer that efficiently compacts nucleic acids to form a stable complex called a polyplex. Since polyplex is taken up into cells by endocytosis, transfection is efficiently performed by using PEI (Non-patent Document 2).

  PEI may be linear PEI or branched PEI, but preferably linear PEI is used. Linear PEI has higher transfection efficiency than branched PEI. The molecular weight of PEI is 10,000-800,000 Da available in view of its transfection efficiency, preferably 10,000-50,000 Da, more preferably 10,000-25,000 Da, and even more preferably about 25,000 Da. Specifically, linear PEI having a molecular weight of about 25,000 Da is preferably used. Chemically modified linear PEI or branched PEI can also be used. PEI is commercially available and can be easily obtained. For example, PEI manufactured by Polysciences, Inc., Warrington, PA, USA can be preferably used.

  The PEI is preferably an acidified PEI solution. In the present specification, the “acidified PEI solution” means a PEI solution prepared by dissolving in an acidic solvent. On the other hand, the “neutralized PEI solution” means a PEI solution prepared by being dissolved in a neutral solvent. The acidity of the acidified PEI solution is preferably pH 0 to pH 2.0, more preferably pH 0.5 to pH 2.0. If the acidity is outside this range, the transfection activity of PEI decreases. As the acidic solvent, any solvent can be used as long as it is a solvent that can maintain the acidity of the PEI solution in the above range and does not adversely affect the transfection activity of PEI. In addition to inorganic acids such as HCl, which may be abbreviated as HCl, acidic solvents such as organic acids having a pH in an acidic region such as an aqueous glycine hydrochloride solution can be exemplified. More specifically, examples of the acidic solvent include 0.01N to 0.5N hydrochloric acid, preferably 0.2N hydrochloric acid.

  The acidified PEI solution is prepared by dissolving PEI powder in an acidic solvent immediately after purchase. Since PEI is easily oxidized by air, it is unstable even in a powdered state, and transfection activity is reduced during storage even when stored in a powdered state at −80 ° C. after opening. Therefore, the purchased PEI powder is preferably dissolved in an acidic solvent immediately after opening. In order to maintain stability during storage, the PEI concentration is preferably dissolved in an acidic solvent at a concentration of 1 mg / ml or more.

  To date, many reports have been made on the success of transfection using a neutralized PEI solution (Non-patent document 2; Non-patent document 6; Non-patent document 12; Pham, PLet al., (2003) Large scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells: Peptone additives improve cell growth and transfection efficiency.Biotechnol. Bioeng. 84, 332-342.; Ehrhardt, C. effective transfection reagent. Signal Transduction 6, 179-184.).

  In practice, however, an unexpected decrease in its transfection activity is often observed during use of PEI. That is, the neutralized PEI solution can be considered unstable.

  On the other hand, the acidified PEI solution almost completely retained its transfection activity even after long-term storage. Specifically, when PEI was dissolved in 0.2N HCl (indicating a pH of about 1), transfection activity was fully maintained for at least 6 months during storage at 4 ° C or -80 ° C. . More specifically, the transfection activity immediately after preparation showed the maximum activity, but the activity was retained without being inactivated even after storage for 6 months. In contrast, when PEI was dissolved in HBS (pH 7.4), transfection activity was dramatically reduced during the 1 month storage period at 4 ° C or -80 ° C. Specifically, assuming that the transfection activity immediately after preparation was 100%, the activity was 0% after storage for 1 month, and the activity was completely inactivated. Furthermore, dissolution of PEI in ethanol dramatically reduced its transfection activity within a few weeks during storage at 4 ° C. Specifically, assuming that the transfection activity at the time of preparation was 100%, the activity was 0% after 2-3 weeks storage, and the activity was completely inactivated.

  In the acidified PEI solution, since the loan pairs of nitrogen atoms of all imino groups are protonated and are not oxidized by air, it is considered that the decrease in transfection activity during storage can be prevented.

  The method for introducing a nucleic acid into a cell according to the present invention is characterized in that PEI and nucleic acid are mixed under acidic conditions, and the cell is treated with the obtained mixture. By mixing PEI and nucleic acid, the nucleic acid is efficiently condensed by PEI, and a stable complex called a polyplex composed of PEI and nucleic acid is formed. Since polyplex is taken up into cells by endocytosis, transfection can be achieved efficiently.

  The acidified PEI solution is preferably used in the method for introducing a nucleic acid into cells according to the present invention.

  “Acid condition” means that the condition is pH 3.5-4.5, preferably pH 4.0. Mixing of PEI and nucleic acid under acidic conditions can be performed, for example, by adding both PEI and nucleic acid to an acidic solvent. Alternatively, PEI and nucleic acid can be separately added to an acidic solvent and then mixed. Hereinafter, in the present specification, a medium used for nucleic acid condensation by mixing PEI and nucleic acid may be referred to as a condensed medium. Any condensate can be used as long as it does not adversely affect cells as a pH 3.5-4.5 buffer, such as lactate buffered saline (LBS), acetate buffered saline, and phosphate buffered saline. Saline can be used. More specifically, as the condensing medium, lactate buffered saline (LBS) having a pH of 4.0 can be exemplified.

  Under conditions of pH 3.5-4.5, preferably pH 4.0, PEI has a sufficiently positive charge, and the phosphate group of nucleic acid also has a negative charge, so the electrical association between PEI and nucleic acid works strongly, As a result, it is considered that a strong polyplex is formed. Under neutral conditions, the nucleic acid has a sufficiently negative charge, but the PEI has a weak positive charge, and under acidic conditions higher than pH 3.5, the nucleic acid does not have a negative charge. It becomes insufficient. If the nucleic acid does not condense to form a polyplex, it will not be taken up by the cell. If it is not a strong polyplex, it will be taken up by the cell and then transported to the lysosome, where it will be degraded by the nuclease. Therefore, the mixing of PEI and nucleic acid is carried out under conditions of pH 3.5-4.5, preferably pH 4.0. The polyplex formed by PEI and nucleic acid has the property of easily leaking into the cytoplasm after being transported to the lysosome, and the leaked polyplex is transported into the nucleus. In the nucleus, the nucleic acid dissociates from the polyplex, returns to a relaxed structure, and performs its function.

  Treatment of cells with a mixture of PEI and nucleic acid can be performed by adding the mixture to a cell solution or a solution containing a tissue derived from a living body. The cells contained in the cell solution may be adherent cells or floating cells.

  The treatment time of the cells with the mixture of PEI and nucleic acid is not particularly limited as long as high transfection efficiency can be obtained, and can be appropriately set by simple repeated experiments. Long-term treatment increases the proportion of dead cells due to the effects of PEI cytotoxicity, thus reducing transfection efficiency. Specifically, the cell treatment time is preferably 8 to 24 hours, more preferably 8 to 12 hours, and even more preferably 8 hours.

  In order to avoid cytotoxicity due to PEI, it is preferable to replace the culture medium of the cells with a fresh culture medium after treatment of the cells with a mixture of PEI and nucleic acid. Changing the culture medium after transfection can minimize the toxicity of PEI without sacrificing high transfection efficiency. If the amount of polyethyleneimine taken up by the cells does not become excessive, it is considered that they are metabolized and nontoxic.

  The mixing ratio of PEI and nucleic acid is not particularly limited as long as high transfection efficiency can be obtained, and can be appropriately set by simple repeated experiments. The appropriate mixing ratio is considered to vary depending on the molecular weight of PEI. For example, when PEI having a molecular weight of around 25,000 Da is used, the mixing ratio of PEI and nucleic acid is preferably in the range of 4: 1 to 8: 1 by weight. If the weight ratio is outside this range, the transfection efficiency of PEI decreases. Furthermore, when the weight ratio is 8: 1 or more, cytotoxicity due to an excessive amount of PEI increases. When PEI is an acidified PEI solution, the weight ratio of PEI and nucleic acid is represented by the weight ratio of PEI and nucleic acid contained in the acidified PEI solution. In addition, when PEI having a molecular weight of about 25,000 Da is used for transfection of cells sensitive to PEI toxicity, it is preferable to reduce the mixing ratio of PEI and nucleic acid to about 1.25: 1 at the maximum.

The method for introducing a nucleic acid into a cell according to the present invention preferably uses a PEI solution prepared by dissolving linear PEI having a molecular weight of about 25,000 in a 0.2N hydrochloric acid solution, and includes the following steps:
(1) A step of mixing PEI and nucleic acid under acidic conditions within a weight ratio of 4: 1 to 8: 1 and within a range of pH 3.5 to 4.5.
(2) treating the cells with the resulting mixture, and (3) replacing the cell culture medium with fresh culture medium,

  A specific example of the method for introducing a nucleic acid into a cell according to the present invention is shown in FIG.

  The method for introducing a nucleic acid into a cell according to the present invention can be used for either transient nucleic acid introduction into a cell or stable nucleic acid introduction into a cell.

  Furthermore, the method for introducing a nucleic acid into a cell according to the present invention can be applied to a cell that is highly sensitive to PEI and easily affected by the cytotoxicity of PEI. When such cells are transfected by this method, the PEI / nucleic acid mixture ratio is reduced more than usual, and the cells are treated with the PEI / nucleic acid mixture multiple times to reduce the effects of PEI cytotoxicity. Can be reduced, and high transfection efficiency can be obtained.

  In general, most of the floating cultured cells do not have sufficient nucleic acid introduced by using chemical transfection reagents. Therefore, nucleic acid introduction is usually carried out by electroporation. Nucleic acid can be introduced by applying this method to cells. Compared with electroporation, this method is useful because it does not require special equipment and can be performed using a small amount of cells.

  The method for introducing a nucleic acid into a cell according to the present invention has a transfection efficiency of about 2 to about 7 times higher than that of a conventional method for introducing a nucleic acid into a cell using neutralized PEI. Was low. Furthermore, the method is less expensive and cost effective compared to non-viral transfection methods that use other reagents.

  The acidified PEI solution can be used as a reagent for introducing nucleic acid into cells. That is, according to the present invention, there can be provided a reagent for introducing a nucleic acid into a cell comprising a PEI solution, wherein the pH of the PEI solution is from 0.5 to 2.0. Preferably, the reagent for introducing nucleic acid into cells according to the present invention comprises a PEI solution prepared by dissolving linear PEI having a molecular weight of about 25,000 in a 0.2N hydrochloric acid solution.

  The acidified PEI solution can be provided as a reagent kit containing the solution in individually packaged form. That is, according to the present invention, a reagent kit containing a PEI solution, wherein the pH of the PEI solution is 0.5 to 2.0, can be provided. Preferably, the reagent kit according to the present invention includes a PEI solution prepared by dissolving linear PEI having a molecular weight of about 25,000 in a 0.2N hydrochloric acid solution. This reagent kit can be preferably used in the method for introducing a nucleic acid into a cell according to the present invention.

  As described above, acidification of PEI can maintain its transfection activity for a long time in introducing DNA into various types of cell lines, and achieve high transfection efficiency and minimal toxicity. In particular, the cost of testing with acidified PEI is dramatically reduced to about 1: 10000 compared to various commercial reagents. Thus, acidified PEI achieves cost-effective and highly efficient transfection.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated more concretely, this invention is not limited at all by the Example shown below.

  First, materials and methods used in the following examples are briefly described.

1. Reagents and plasmids Linear PEI (molecular weight 25,000) was purchased from Polysciences, Inc. PEI powder was dissolved in 0.2N HCl at a concentration of 5 mg / ml and a portion was stored at 4 ° C or -80 ° C. HEPES buffered saline (hereinafter abbreviated as HBS: 20 mM HEPES-KOH, pH 7.4, and 150 mM NaCl) and lactate buffered saline (hereinafter abbreviated as LBS: 20 mM sodium lactate, pH 3.5, 4.0) Or 4.5 and 150 mM NaCl). TransIT-LT1 transfection reagent was purchased from Mirus Bio LLC. A vector pcDNA4-TO-EGFP encoding an enhanced green flulorescent protein (hereinafter abbreviated as EFGP) was constructed by a previously reported method using a pcDNA4 / TO vector (Invitrogen) (Nakayama). , Y. et al., (2006) Involvement of the N-terminal unique domain of Chk tyrosine kinase in Chk-induced tyrosine phosphorylation in the nucleus. Exp. Cell Res. 312, 2252-2263.). The pCAG vector encoding CD25 (pCAG / TetR-IRES-CD25) was constructed by a previously reported method (Miyazaki, J. et al., (1989) Expression vector system based on the chicken beta-actin promoter directs efficient production of interleukin-5. Gene 79, 269-277.).

2. Cell Lines Human epithelial HeLa and HeLa S3 cells (Japanese Collection of Research Bioresources, Osaka) contain Iscove's modified Dulbecco's medium containing 5% fetal bovine serum (FBS) and 5% bovine serum (BS), respectively. ; IMDM). Human megakaryocyte Dami cells were maintained by suspension culture in IMDM containing 10% horse serum as previously reported (Greenberg, SM et al., (1988) Characterization of a new megakaryocytic cell line: The Blood 72, 1968-1977 .; Hirao, A. et al., (1998) Overexpression of C-terminal Src kinase homologous kinase suppresses activation of Lyn tyrosine kinase required for VLA5-mediated Dami cell spreading. J. Biol. Chem. 273, 10004-10010 .; Sato, I. et al., (2009) Differential trafficking of Src, Lyn, Yes and Fyn is specified by the state of palmitoylation in the SH4 domain. J. Cell Sci. 122, 965 -975.), And attached to the culture dish by culturing for 2-3 days in IMDM containing 2.5% FBS and 2.5% horse serum.

3. Examination of influence of DNA condensation pH and PEI / DNA ratio on transfection efficiency
HeLa cells were seeded in a 35 mm culture dish at 1 × 10 ⁵ cells per dish and cultured for 1-2 days. Prior to transfection, the culture medium was replaced with fresh serum-containing medium. To form a PEI / DNA polyplex, 1 μg of pcDNA4-TO-EGFP was mixed with PEI at a PEI / DNA ratio described below in 100 μl of LBS or HBS, and the mixture was left at room temperature for 20 minutes. In early experiments, 1 μg pcDNA4-TO-EGFP and 5 μg PEI were mixed directly in 100 μl LBS, but in subsequent experiments, 1 μg pcDNA4-TO-EGFP and 5 μg PEI were each separated in 50 μl LBS. Diluted to mix. The resulting PEI / DNA polyplex was diluted with 500 μl of pre-warmed serum-free IMDM and added dropwise to each culture dish. 24 hours after transfection, cells were detached by gently pipetting in phosphate buffered saline (PBS) containing 0.05% EDTA. After centrifugation, resuspend cells in PBS containing 3% FBS and 1 μg / ml propidium iodide (PI), and use EGFP fluorescence to verify protein expression and PI staining to verify dead cells The analysis was performed by flow cytometry using a MoFlo cell sorter (Beckman Coulter). Transfected cells grown in culture dishes were observed under a Zeiss LSM 5 Pascal deconvolution microscope (Zeiss LSM 5 Pascal deconvolution microscope, Carl Zeiss) 24 hours after transfection.

4. Examination of transfection of cell lines sensitive to PEI toxicity
HCT116 cells are sensitive to the toxicity of transfection reagents. In order to reduce the toxicity of PEI and increase transfection efficiency, a sequential transfection method was developed. Specifically, DNA / PEI polyplex was prepared using 7.5 μg PEI and 6 μg pCAG / TetR-IRES-CD25, added dropwise to each 35 mm culture dish, and after 8 hours incubation the culture medium Was replaced with fresh serum-containing medium. For further subsequent transfections, freshly prepared DNA / PEI polyplexes are added to the cell culture as indicated in the first transfection, and after an additional 8 hours of incubation, the medium is reconstituted with fresh serum-containing medium. Exchanged. Cells were further cultured for 16 hours, stained with anti-CD25 antibody, and cell surface CD25 expression was analyzed as previously reported by flow cytometry using a MoFlo cell sorter (Nakayama, Y. et al., ( 2005) Multi-lobulation of the nucleus in prolonged S phase by nuclear expression of Chk tyrosine kinase. Exp. Cell Res. 304, 570-581.

5. Creation of cell clones that stably express EGFP
HeLa S3 cells were seeded in a 60 mm culture dish at 3 × 10 ⁴ cells per dish and cultured for 1 day. To form the PEI / DNA polyplex, 97.5 μg PEI and 15 μg pcDNA4-TO-EGFP are each separately diluted in 50 μl LBS (pH 3.5), and the dilutions are mixed and transferred as above. Used for the effect. Cells grown in culture dishes 24 hours after transfection were divided into 4 dishes, and selection of stable transfected cells was performed 48 hours after transfection in 250 μg / ml Zeocin (Invitrogen). Conducted from the eyes.

  The effects of acidic pH on PEI storage stability, PEI / DNA polyplex formation, and PEI toxicity were investigated.

  To date, there have been many reports of successful transfection with neutralized PEI solutions. However, in fact, an unexpected decrease in its transfection activity was often observed during use of PEI. That is, it was estimated that the neutralized PEI solution was unstable. Therefore, the effect of acidic pH on the stability of PEI during storage was examined.

  When PEI was dissolved in HBS (pH 7.4), transfection activity was dramatically reduced during the 1 month storage period at 4 ° C or -80 ° C. Specifically, the transfection activity immediately after the preparation was completely inactivated with the activity being 0% after storage for 1 month, assuming that the transfection activity was 100%. Furthermore, dissolution of PEI in ethanol dramatically reduced its transfection activity within a few weeks during storage at 4 ° C. Specifically, assuming that the transfection activity at the time of preparation was 100%, the activity was 0% after 2-3 weeks storage, and the activity was completely inactivated. Here, the transfection activity was evaluated by the ratio of the number of EGFP gene-expressing cells to the total number of cells when a vector containing the EGFP gene was introduced into COS-1 cells.

  In contrast, when PEI was dissolved in 0.2N HCl (indicating a pH of about 1), transfection activity was fully maintained for at least 6 months during storage at 4 ° C or -80 ° C. Specifically, assuming that the transfection activity immediately after preparation was 100%, it was 100% even after storage for 6 months.

  This suggests that a protective effect against air oxidation can be obtained by fully protonating the amino group of PEI in 0.2N HCl. On the other hand, the potential of PEI powder for transfection was greatly lost during storage at 4 ° C or -80 ° C once the container was opened. It can be considered that PEI begins to oxidize by the internal air, thereby causing a decrease in transfection activity.

  Next, the effect of pH on polyplex formation was examined. That is, polyplex formation was performed using acidic and neutral condensate media, and transfection efficiency was compared. Specifically, PEI and pcDNA4-TO-EGFP were mixed in HBS (pH 7.4) or LBS (pH 3.5) so that the PEI / DNA ratio was 5 (μg / μg), and the obtained PEI / DNA HeLa cells were transiently transfected with polyplex. EGFP expression was analyzed by flow cytometry 24 hours after transfection. Transfection efficiency was assessed by counting the number of cells expressing EGFP at high and low to high levels.

  As a result, the number of cells expressing EGFP when LBS (pH 3.5) was used as the condensation medium was larger than that when HBS (pH 7.4) was used (FIG. 1-A). Similar results were obtained by fluorescence microscopy (data not shown). At physiological pH, where only the PEI fraction forms a complex with DNA, every six amino groups are protonated, half of the amino groups are protonated at pH 5, and 80% amino at pH 3. The group is reported to be protonated (Suh, J. et al., (1994) Ionization of poly (ethylenimine) and poly (allylamine) at various pH's. Bioorg. Chem. 22, 318-327 .; Clamme, JP et al., (2003) Monitoring of the formation and dissociation of polyethylenimine / DNA complexes by two photon fluorescence correlation spectroscopy. Biophys. J. 84, 1960-1968 .; Menzel, H. et al., (2003) Chemical properties of polyamines with relevance to the biomineralization of silica. Chem. Commun. 2003, 2994-2995.). From this, it can be considered that the increase in positive charge of PEI promotes DNA / PEI polyplex formation.

  Furthermore, the effect of acidic pH on the toxicity of PEI was investigated. HeLa cells transiently transfected with polyplexes formed in HBS (pH 7.4) or LBS (pH 3.5) were stained with PI, and dead cells stained with PI were analyzed by flow cytometry.

  As a result, in LBS, the number of dead cells stained with PI was smaller than that in HBS (FIG. 1-B). This suggests that the use of LBS can alleviate the toxicity of PEI. A significant part of the toxicity comes from free PEI molecules that do not complex with DNA (Godbey, WT et al., (2001) Poly (ethylenimine) -mediated gene delivery affects endothelial cell function and viability. Biomaterials 22, 471- Moghimi, SM et al., (2005) A two-stage poly (ethylenimine) -mediated cytotoxicity: Implications for gene transfer / therapy. Mol. Ther. 11, 990-995.). Therefore, it can be considered that enhancement of polyplex formation contributes to reduction of toxicity.

  From the above results, it was found that storage with an acidified PEI solution is preferable in order to maintain stability during the storage period of PEI and obtain high transfection activity.

  Further, from the above results, it was found that adopting an acidic condensed medium instead of a neutral condensed medium as a condensing medium at the time of polyplex formation improves transfection efficiency and mitigates toxicity.

  Optimization of PEI / DNA ratio and condensation pH during polyplex formation were investigated.

  The transfection efficiency of PEI depends on the ratio of PEI to DNA (Non-patent Document 2). Therefore, PEI and DNA were mixed in LBS (pH 3.5) at various PEI / DNA ratios ranging from 1 to 10, and transfection efficiency was compared using the resulting polyplex. Specifically, PEI and pcDNA4-TO-EGFP were mixed at various PEI / DNA ratios in LBS (pH 3.5), and HeLa cells were transiently transfected with the obtained PEI / DNA polyplex. . Cells were stained with PI for detection of dead cells 24 hours after transfection, and EGFP expression and toxicity were analyzed by flow cytometry.

  The number of EGFP expressing cells increased significantly when the PEI / DNA ratio ranged from 4 to 8 (FIGS. 2-A and 2-B). The optimized PEI / DNA ratio was consistent with 40-50, the ratio of nitrogen in PEI to NP in DNA (NP ratio) (Figure 2-A). However, it was recommended to use linear low molecular weight PEI at a NP ratio of 6 in neutral condensate (Non-Patent Documents 4 and 8; Ferrari, S. et al., (1997) ExGen 500 is an efficient vector. for gene delivery to lung epithelial cells in vitro and in vivo. Gene Ther. 4, 1100-1106. The number of dead cells stained with PI increased when the PEI / DNA ratio was 10 (FIG. 2-A). Excess free PEI molecules contribute to efficient transfection, but suppressed their transfection efficiency due to their high toxicity (Fig. 2-A; Clamme, JP et al., (2003) Monitoring of the formation and dissociation of polyethylenimine / DNA complexes by two photon fluorescence correlation spectroscopy. Biophys. J. 84, 1960-1968 .; Ira, MY et al., (2003) DNA vector polyethyleneimine affects cell pH and membrane potential: A time-resolved fluorescence microscopy study. J. Fluoresc. 13, 339-347.). Larger size vectors (> 10 kbp) are preferred (data not shown) for efficient transfection when the PEI / DNA ratio is low (˜3) due to excess free PEI.

  Next, the effect of DNA condensation pH on transfection efficiency was examined. Specifically, PEI and pcDNA4-TO-EGFP were mixed in HBS (pH 7.4) or LBS (pH 3.5, 4.0, 4.5) to a ratio of 5 (μg / μg) and obtained HeLa cells were transiently transfected with PEI / DNA polyplex.

  As a result, the number of EGFP-expressing cells increased 2-fold to 6-fold when using LBS at pH 3.5, pH 4.0, and pH 4.5 as the condensing medium compared to using HBS at pH 7.4. (Fig. 2-C). In addition, pH 4.0 LBS was found to be the most efficient condensing medium among the condensing media tested (FIG. 2-C).

  Therefore, to achieve high PEI-mediated transfection efficiency, it is recommended to use a condensing medium, preferably about pH 3.5 to about 4.5, more preferably about pH 4.0, such as LBS at such pH.

  PEI was used to examine the transfection of various cells. As a result, HeLa, HeLa S3, A431, COS-1, MCF7, NIH3T3, HCT116 and HEK293 cells could be transfected using the PEI / DNA polyplex formed with LBS (pH 4.0). The typical results are shown below.

  First, COS-1 and HeLa S3 cells were prepared using a polyplex formed by mixing 5 μg pcDNA4-TO-EGFP and 25 μg PEI (PEI / DNA ratio 5) in LBS (pH 4.0). Transfected transiently and after 12 hours of culture the medium was replaced with fresh serum-containing medium. EGFP expression was then measured to assess transfection efficiency. As a result, the transfection efficiency of COS-1 and HeLa S3 cells was ˜80% and ˜60%, respectively (FIG. 3-A).

  Next, using COS-1 cells, transfection efficiency was compared between PEI dissolved in LBS (pH 4.0) and the commercially available transfection reagent TransIT-LT1. Specifically, cells were either polyplex formed by mixing 1 μg pcDNA4-TO-EGFP and 5 μg PEI (PEI / DNA ratio 5) in LBS (pH 4.0), or optimal amount of TransIT Transient transfection with -LT1. EGFP expression was then measured to assess transfection efficiency. As a result, it was revealed that the transfection efficiency by PEI is comparable to the transfection efficiency by TransIT-LT1 (Fig. 3-B).

  Next, transfection of suspension cells by PEI was examined using human megakaryocyte Dami cells, which are suspension culture cells. Since many suspension culture cells are not fully transfected with chemical transfection reagents, transfection is usually performed by electroporation using special equipment and large numbers of suspension cells. Such a method causes severe cell death. Therefore, it was investigated whether buoyant cells could be transfected by a transfection method using PEI / DNA polyplexes formed under acidic conditions using acidified PEI. Dami cells were found to adhere to the culture dish when grown in medium supplemented with FBS and horse serum (see materials and methods above), so that the Dami cells cultured in suspension were attached to the culture dish, and Transient transfection was performed using a polyplex formed by mixing 4 μg of pcDNA4-TO-EGFP and 26 μg of PEI (PEI / DNA ratio is 6) in LBS (pH 4.0). As a result, Dami cells can be transfected with PEI / DNA polyplexes formed with LBS (pH 4.0), and the transfection rate is 3-5%, which is equivalent to the transfection rate by electroporation. It became clear that it was comparable (Figure 3-C). Since the transfection method using PEI does not require any special equipment and can be applied even to a small amount of cells, it is useful for transfection of suspension cells compared to the transfection method by electroporation.

  In addition, transfection using PEI / DNA polyplexes formed under acidic conditions using acidified PEI was examined for cells that are highly sensitive to contact with PEI and therefore prone to cell aggregation and cell death. . HCT116 cells were used as cells sensitive to contact with PEI. Then, the PEI / DNA ratio was reduced to 1.25 in order to reduce the toxicity of PEI, and the transfection operation was repeated twice in order to increase the transfection efficiency. Specifically, transiently transfer once or twice using polyplex formed by mixing 6 μg pCAG / TetR-IRES-CD25 and 7.5 μg PEI in LBS (pH 4.0). And staining with anti-CD25 antibody, followed by analysis of CD25 expression on a cell sorter. As a result, a transfection efficiency of about 30% was obtained by the continuous transfection method (FIG. 3-D).

Next, high transfection efficiency and low toxicity were achieved by the transfection method using PEI / DNA polyplexes formed under acidic conditions using acidified PEI, making stable transfection cell clones easy We examined whether to be done. Specifically, stable transfection of HeLa S3 cells was performed using a polyplex formed by mixing pcDNA4-TO-EGFP and PEI in LBS. Zeocin resistant colonies were then cloned in 2 weeks. As a result, more than 200 colonies derived from a single cell and stably expressing EGFP were obtained from 3 × 10 ⁴ HeLa S3 cells (FIG. 3-E). In addition, stable transfected cell clones could be created from PEI-sensitive HCT116 cells (data not shown).

  Furthermore, this transfection method using PEI enabled efficient transfection of various cell lines such as HeLa, COS-1 and Dami cells with shRNA vectors for gene knockdown (data not shown) ).

  According to the present invention, a method for introducing a nucleic acid into a cell that achieves high transfection efficiency and minimal toxicity can be provided. Furthermore, the method according to the present invention is cost effective compared to conventional methods. In addition, the acidified PEI solution provided by the present invention is stable and can maintain its transfection activity for a long period of time, so that high transfection efficiency and minimal toxicity can be achieved in introducing nucleic acids into cells. .

  Introduction of nucleic acids into cells is essential for basic research in molecular cell biology as well as medical applications such as gene therapy. Therefore, the present invention is useful to contribute widely from the basic science field to the pharmaceutical research and development field.

Claims (6)

A method for introducing a nucleic acid into a cell, comprising the following steps:
(1) mixing polyethyleneimine (PEI) and nucleic acid under acidic conditions of pH 3.5 to pH 4.5, and (2) treating cells with the resulting mixture,
Here, the PEI is a linear PEI having a molecular weight of 25,000 . The method for introducing a nucleic acid into a cell according to claim 1, further comprising the following step following the step (2):
(3) A step of replacing the cell culture medium with a fresh culture medium. 3. The method for introducing a nucleic acid into a cell according to claim 1, wherein the weight ratio of polyethyleneimine (PEI) to the nucleic acid is 4: 1 to 8: 1. 4. The method for introducing a nucleic acid into a cell according to claim 1, wherein the polyethyleneimine (PEI) is a solution having a pH of 0.5 to pH 2.0 (PEI solution). 4. The method for introducing a nucleic acid into a cell according to claim 1, wherein the polyethyleneimine (PEI) is a solution prepared by dissolving PEI in a 0.2N hydrochloric acid solution. A method for introducing a nucleic acid into a cell, comprising the following steps:
(1) A step of mixing polyethyleneimine (PEI) and nucleic acid under acidic conditions of pH 3.5 to pH 4.5,
(2) treating the cells with the resulting mixture, and (3) replacing the cell culture medium with fresh culture medium,
Here, the PEI is a solution prepared by dissolving linear PEI having a molecular weight of 25,000 in 0.2N hydrochloric acid solution, and the weight ratio of PEI to nucleic acid is 4: 1 to 8: 1.